Microglia derived from pluripotent stem cells and methods of making and using the same

ABSTRACT

The present invention provides methods and compositions for the generation of microglial progenitor cells and microglial cells from pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells. The present invention also provides cells produced using such methods, and both methods of treatment and methods of drug screening that use such cells. Also provided are various tissue culture media, tissue culture media supplements, and kits useful for the generation of human microglial progenitor cells and human microglial cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 62/303,301, filed on Mar. 3, 2016 and U.S.Provisional Patent Application No. 62/410,645, filed on Oct. 20, 2016,the contents of each which are hereby incorporated by

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numberAG046170 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

INCORPORATION BY REFERENCE

For the purpose of only those jurisdictions that permit incorporation byreference, all of the references cited in this disclosure are herebyincorporated by reference in their entireties (numbers in parentheses orin superscript following text in this patent disclosure refer to thenumbered references provided in the “Reference List” section of thispatent specification). In addition, any manufacturers' instructions orcatalogues for any products cited or mentioned herein are incorporatedby reference. Documents incorporated by reference into this text, or anyteachings therein, can be used in the practice of the present invention.

BACKGROUND OF THE INVENTION

Microglia are the resident, tissue-specific macrophages of the brain.They perform several critical roles in the development and maintenanceof the central nervous system (CNS). Microglia arise from primitiveCD45+CX3CR1− myeloid progenitors in the yolk sac that differentiate toCD45+CX3CR1+ microglial progenitors and invade the developing brainbefore the emergence of definitive hematopoiesis. In the healthy, adultbrain with an intact blood brain barrier, microglia persist as along-lived, self-sustained population that is not replenished bycirculating bone marrow-derived cells. Highly branched microglia cells,defined as “resting”, are in reality highly active as their processescontinuously move to examine the brain for homeostatic disruptions.

Microglia use phagocytosis to eliminate pathogens and/or damaged or deadcells, and remove toxic molecules, cellular debris, and/or proteindeposits, thus attenuating inflammation and promoting tissueregeneration and repair. During development, microglia promote migrationand differentiation of neural progenitors, neurogenesis, andoligodendrogenesis, and regulate synaptogenesis and synaptic plasticitythrough pruning. Microglia can also contribute to pathological braininflammation and disruption of the blood-brain barrier by releasingcytokines and neurotoxic molecules. Dysfunctional microglia have beenlinked to amyotrophic lateral sclerosis (ALS) and Alzheimer's disease(AD). Chronic activation of microglia cells is a possible trigger to theprogression of multiple sclerosis (MS) and Parkinson's disease, anddefective phagocytosis and synaptic pruning have been implicated in thepathogenesis of schizophrenia and autism spectrum disorders. Otherdiseases determined to have microglial involvement based on animalstudies include Rett syndrome, diffuse leukoenchephalopathy withspheroids (such as hereditary diffuse leukoenchephalopathy with axonalspheroids), and frontotemporal lobar degeneration (FTLD), such asfamilial FTLD.

Most of our knowledge regarding microglia derives from rodent studies.However, there are major differences between rodent and human microglialcells, such as in their proliferation rates, adhesive properties, andexpression of critical receptors. Consistent with these differences,protocols developed previously for the differentiation of rodentpluripotent stem cells into microglia have not been effective with humanpluripotent stem cells.

Direct analysis of primary human microglial cells has been severelyhampered by the limited availability of human brain specimens,especially from healthy individuals. While the development of the humanpluripotent stem cell (PSC) field has enabled the generation of manydifferent differentiated cell types from human pluripotent stem cells,there has remained a need in the art for efficient and reproduciblemethods for the generation of microglial cells from such human stemcells. The present invention addresses this need.

SUMMARY OF THE INVENTION

Microglia, the immune cells of the brain, are crucial to the properdevelopment and maintenance of the central nervous system, and areinvolved in numerous neurological diseases and disorders. The presentinvention provides various new and improved methods for the generationof human microglia from pluripotent stem cells. Using chemically definedmedia, the methods of the present invention are used to generatemicroglial progenitors expressing CD14 and/or CX3CR1 from both embryonicstem cells (ES cells or ESCs) and induced pluripotent stem cells (iPScells or IPSCs). Such microglial progenitors typically appear withinaround 25-30 days and continue to be produced for around 20-25days—until around day 50. The methods provided herein also enable thefurther differentiation of such microglial progenitors—resulting in thegeneration of ramified microglia that have highly motile processes,express many typical microglial markers, release cytokines, havephagocytotic activity, and respond to ADP by producing intracellularCa²⁺ transients. These methods are highly reproducible across differentpluripotent stem cell (PSC) lines.

Some of the main aspects of the present invention are summarized below.Additional aspects are described in the Detailed Description of theInvention, Examples, Figures, and Claims sections of this disclosure.The description in each section of this disclosure is intended to beread in conjunction with the other sections. Furthermore, the variousembodiments described in each section of this disclosure can be combinedin various different ways, and all such combinations are intended tofall within the scope of the invention.

In some embodiments the present invention provides methods forgenerating microglial cells from pluripotent stem cells. In some suchembodiments the pluripotent stem cells are from any mammalian species.In some embodiments the pluripotent stem cells are human pluripotentstem cells—resulting in the generation of human microglial cells. Insome embodiments the pluripotent stem cells are either inducedpluripotent stem cells (“iPS cells” or “iPSCs”), or embryonic stem cells(“ES cells” or “ESCs”). Such methods involve two main steps. In thefirst step, the pluripotent stem cells are cultured under conditionsthat induce myeloid differentiation, leading to the generation of CD14+and/or CX3CR1+ microglial progenitor cells. In the second step, theCD14+ and/or CX3CR1+ microglial progenitor cells are differentiated intomicroglial cells. In some embodiments the CD14+ and/or CX3CR1+microglial progenitor cells are differentiated into microglial cells byculturing them in either (i) a first microglial differentiation mediumcomprising IL-34, or (ii) a second microglial differentiation mediumcomprising M-CSF. In some embodiments the first microglialdifferentiation medium also comprises GM-CSF. In some embodiments thesecond microglial differentiation medium also comprises one or morefactors selected from the group consisting of GM-CSF, NGF-β and CCL2.For example, in some embodiments the second microglial differentiationmedium also comprises each of GM-CSF, NGF-β and CCL2. In someembodiments the CD14+ and/or CX3CR1+ microglial progenitor cells arecontacted with the first or second microglial differentiation medium foraround 15 days. In some embodiments, the CD14+ and/or CX3CR1+ microglialprogenitor cells generated in the first step are isolated beforeproceeding to the second step. In some embodiments the microglialprogenitor cells are isolated at around the time that CD14 expressionpeaks.

In some alternative embodiments, the present invention provides methodsfor generating microglial progenitor cells from pluripotent stem cells.Such methods involve performing the first of the two main stepsdescribed above, including, optionally, isolating the resulting CD14+and/or CX3CR1+ microglial progenitor cells.

In other alternative embodiments, the present invention provides methodsfor generating microglial cells from microglial progenitor cells. Suchmethods involve performing the second of the two main steps describedabove.

In some of the embodiments of the present invention that involveculturing pluripotent stem cells under conditions that induce myeloiddifferentiation (leading to the generation of CD14+ and/or CX3CR1+microglial progenitor cells), a multi-step process is used in which thecells are cultured with different combinations of cytokines and tissueculture media at each stage. Steps in these multi-step processes mayresult in inducing differentiation of pluripotent stem cells intoprimitive hemangioblasts, and/or inducing differentiation of primitivehemangioblasts into myeloid progenitors, and/or inducing differentiationof myeloid progenitors into microglial progenitors. As defined by theircell surface marker profiles, in some embodiments such multi-stepprocesses may result in first inducing differentiation of thepluripotent stem cells into KDR+CD235a+ cells, then into CD45+CX3CR1−cells, and then into CD45+CX3CR1+ CD14+ cells.

In some embodiments, the methods provided herein for the generation ofCD14+ and/or CX3CR1+ microglial progenitor cells from pluripotent stemcells comprise performing one or more of the following four steps:First, contacting a cell culture with a first composition comprisingBMP4 in a culture medium, wherein when the cell culture is initiallycontacted with the first composition the cell culture comprisespluripotent stem cells; Second, contacting the cell culture with asecond composition comprising one or more of bFGF, SCF, and VEGFA (forexample each of bFGF, SCF, and VEGFA) in a hematopoietic cell medium;Third, contacting the cell culture with a third composition comprisingone or more of SCF, IL-3, TPO, M-CSF, and FLT3 ligand (for example eachof SCF, IL-3, TPO, M-CSF, and FLT3 ligand) in a hematopoietic cellmedium; and Fourth contacting the cell culture with a fourth compositioncomprising one or more of M-CSF, FLT3 ligand, and GM-CSF (for exampleeach of M-CSF, FLT3 ligand, and GM-CSF) in a hematopoietic cell medium.In some embodiments all of the above four steps are performed in order.In some of such embodiments the medium used for any of these four stepsis a serum free medium. In some of such embodiments the medium used forany of these four steps is a chemically-defined medium. Exemplaryamounts of the agents used in these four compositions are provided inthe Detailed Description and Examples sections of this patentdisclosure.

In the first of the above four steps, in some embodiments a tissueculture medium suitable for maintenance of stem cells is used, while inother embodiments a tissue culture medium suitable for differentiationof stem cells is used. In some embodiments a modified pluripotent stemcell maintenance medium that does not comprise pluripotency factors isused. In some embodiments a medium does not comprise pluripotencyfactors is used. For example, in one embodiment, the medium used in thefirst of the above four steps does not comprise lithium chloride, GABA,pipecolic acid, bFGF or TGFβ1. For example, in one embodiment a modified“mTeSR1” medium that does not contain lithium chloride, GABA, pipecolicacid, bFGF or TGFβ1 is used. In the last three of the above four steps,any suitable hematopoietic cell medium can be used. In one embodimentthe hematopoietic cell medium is “StemPro-34.”

The present invention also provides certain exemplary timings forperforming each of the four steps described above for the generation ofCD14+ and/or CX3CR1+ microglial progenitor cells from pluripotent stemcells. These exemplary timings are described in the DetailedDescription, the Examples and in the Figures. For example, in someembodiments the cell cultures are contacted with the first compositionfor approximately 4 days. In some embodiments the cell cultures arecontacted with the second composition for approximately 2 days. In someembodiments the cell cultures are contacted with the third compositionfor approximately 8 days. In some embodiments the cell cultures arecontacted with the fourth composition for approximately 11 to 36 days,or more.

In some embodiments, when carrying out the methods described above orelsewhere herein for the generation of CD14+ and/or CX3CR1+ microglialprogenitor cells from pluripotent stem cells, instead of discarding thetissue culture supernatant when performing media changes, thesupernatant is saved and the cells present in the supernatant arerecovered and added back to the cell cultures. This is advantageousbecause the inventors have discovered that certain of the key cell typesinduced during the conversion of pluripotent stem cells to microglialprogenitors are found predominantly in the cell supernatants—as opposedto being in the layer of cells that adheres to the cell culture plates.Thus, in some embodiments, when media is changed cells present in theculture supernatant are recovered and added back to the cell cultures.In some embodiments, for media exchanges performed after about day 10,cells present in the culture supernatant are recovered and added back tothe cell cultures. In some embodiments, for media exchanges performedwhen the cells are in contact with the third composition or the fourthcomposition, cells present in the culture supernatant are recovered andadded back to the cell cultures. In some embodiments, for mediaexchanges performed after the emergence of a CD45+CX3CR1− cellpopulation, cells present in the culture supernatant are recovered andadded back to the cell cultures.

In some embodiments the present invention provides microglial cells ormicroglial progenitor cells, such as those produced by the methodsdescribed herein. In some embodiments the present invention provides“substantially pure” populations of such cells.

In some embodiments the present invention provides CD14+ microglialprogenitor cells, such as those produced by the methods provided herein.In some embodiments the present invention provides CX3CR1+ microglialprogenitor cells, such as those produced by the methods provided herein.In some embodiments the present invention provides CD14+ CX3CR1+microglial progenitor cells, such as those produced by the methodsprovided herein. In some embodiments the present invention providesCD14+ CX3CR1+ CD45+ microglial progenitor cells, such as those producedby the methods provided herein.

In some embodiments the present invention provides microglial cells,such as those produced by the methods provided herein, that express oneor more markers selected from the group consisting of CDllb, CDllc,CX3CR1, P2RY12, IBA-1, TMEM119, and CD45.

In some embodiments the present invention provides CDllb+, CDllc+,CX3CR1+ P2RY12+ CD45+ microglial cells, such as those produced by themethods provided herein.

In some embodiments the present invention provides CDllb+, CDllc+,CX3CR1+ P2RY12+ CD45+, IBA-1+, TMEM119+ microglial, such as thoseproduced by the methods provided herein.

In some embodiments the present invention provides microglial cells,such as those produced by the methods provided herein, that expresslower levels of CD11b, OLFML3 and/or TMEM119 than primary microglialcells.

In some embodiments the microglial cells provided herein have a ramifiedmorphology, and/or have phagocytic activity, and/or produceintracellular Ca2+ transients in response to adenosine diphosphate (ADP)exposure, and/or release cytokines, and/or have a transcriptionalprofile similar to that of primary microglial cells.

The microglial cells and microglial progenitor cells generated using themethods of the present invention can be used for any desired purpose,including, but not limited to, in research, in drug screening, in animalmodels (such as models of human disease), in methods of treatment, instudying the effects of their released cytokines (for example on othercell types, such as neurons), in direct or indirect co-cultures withother cell types (e.g. neurons, astrocytes, oligodendrocytes, and/orbrain endothelial cells), in producing conditioned media for culture ofother cell types (e.g. neurons, astrocytes, oligodendrocytes, and/orbrain endothelial cells), in the generation in organ cultures and/or 3dimensional tissue cultures (such as mini-brains or brain organoids),and the like. One of skill in the art will recognize that the microglialcells and microglial progenitor cells described herein, and/or thoseproduced using the methods described herein, can be used for any purposefor which it is, or would be, desirable to use any other microglial cellor microglial progenitor cell.

For example, in some embodiments the present invention provides methodsof treatment and methods of prevention comprising administering amicroglial cell or microglial progenitor cell as described herein, or asproduced using a method described herein, to a subject in need thereof.In some embodiments the subject may have, or be suspected of having, orbe at risk of developing, a disease or disorder associated with a defectin or deficiency of microglial cells or microglial progenitor cells.Such diseases and disorders include, but are not limited to amyotrophiclateral sclerosis (ALS), Alzheimer's disease (AD), multiple sclerosis(MS), Parkinson's disease, Rett syndrome, diffuse leukoenchephalopathywith spheroids (such as Hereditary Diffuse Leukoenchephalopathy withAxonal Spheroids), frontotemporal lobar degeneration (FTLD), such asfamilial FTLD, schizophrenia and autism spectrum disorders. In some suchmethods the microglial cell or microglial progenitor cell used intreatment is generated from an induced pluripotent stem cell derivedfrom the same subject—i.e. it is an autologous cell. In other suchembodiments the microglial cell or microglial progenitor cell used intreatment is generated from a different individual (a donor) of the samespecies, i.e. it is an allogeneic cell. In some embodiments where anallogeneic/donor cell is used, the cell may be derived from a donorindividual having a MHC/HLA type that matches that of the subject.

In other embodiments the present invention provides methods of drugscreening using microglial cells or microglial progenitor cells asdescribed herein, or as produced using the methods described herein,including, but not limited to, high-throughput screening methods. Forexample, in one embodiment the present invention provides a method ofidentifying a compound useful in the treatment or prevention of adisease or disorder associated with a defect in or deficiency ofmicroglial cells or microglial precursor cells, the method comprising:contacting a microglial cell or microglial progenitor cell as describedherein, or as generated using a method described herein, with one ormore candidate compounds, and determining whether any one or more of thecandidate compounds improves the defect in or deficiency of microglialcells or microglial precursor cells. In some embodiments the presentinvention provides screening methods aimed at identifying modulators ofthe P2RY12 G-protein-coupled receptor expressed by the microglial cellsdescribed herein, or expressed by the microglial cells generated usingthe methods described herein. Such methods may comprise contacting suchmicroglial cells with one or more candidate compounds, and determiningwhether any of the candidate compounds modulate the activity of theP2RY12 G-protein-coupled receptor.

In other embodiments the present invention also provides tissue culturemedia, tissue culture media supplements, and various kits useful inperforming the various methods described herein—as described further inthe Detailed Description section of this patent disclosure.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings are forillustration purposes only, and are not intended to limit the scope ofthe present teachings.

FIG. 1 A-B. PSCs differentiate to microglia through myeloid progenitors.(A) Schematic illustration of the major steps of the microglialdifferentiation protocol of the present invention. (B) Kinetics of CD45,CX3CR1 and CD14 expression between day 10 and day 25 in the adherent andsupernatant fractions.

FIG. 2 A-C. Characterization of iPSC-MG. (A) Panel of representativeimages of iPSC-MG in phase contrast and after immunofluorescent labelingfor IBA1, CD11c, TMEM119 and P2RY12. White boxes indicate the areas ofthe magnified insets. Scale bars are 50 μm, 200 μm, 200 μm, 200 μm and200 μm. (B) Flow cytometry plots for typical microglial surface antigensin iPSC-MG. (C) Dot-plot showing the percentage of total cellsexpressing the microglial surface antigens shown in B across 4independent iPSC-MG (depicted as circles) and 2 hMG samples (depicted astriangles). Error bars indicate the mean±SEM.

FIG. 3 A-E. Gene expression, cytokine release profile, and phagocytosisof microglia and macrophages. (A) Hierarchical clustering dendrogram ofthe RNAseq data based on global mRNA expression. Sample distances werecalculated from Pearson's correlation coefficient analysis. (B)Dendrogram showing hierarchical clustering of our RNAseq data and dataobtained from an independent study of human primary CD45+ cells in thebrain (“Myeloid”, GEO: GSE73721). Analysis is based ontranscriptome-wide expression. (C) Graphs showing the expression levelsof the six human microglial signature genes. Samples correspond to datafrom independent studies (GSE73721, GSE85839). (D) Heat-map of thereleased cytokine profiles of 5 independent iPSC-MG runs from 2 lines, 2independent hMG samples and one hMG-SF sample compared to PB-M. Arrowsindicate the 5 proteins upregulated in hMG and M(LPS, IFNγ) only. (E)Representative fluorescent image and flow cytometry histograms showingphagocytosis of YG-labeled microspheres. iPSC: Undifferentiated iPSCsused as negative control.

FIG. 4 A-F. ADP-evoked [Ca2+]i transients in microglia and macrophages.(A) Left panels show five example traces of intracellular Ca2+transients following bath application of ADP in iPSC-MG loaded with theCa2+ indicator Fluo-4/AM. Right panels show time lapse of changes influorescence intensity produced by ADP application. Traces originatefrom cells indicated by regions of interest in right panel. Barsrepresent duration of ADP or ATP application. (B-C) Same data as in A,obtained from primary human microglia (hMG) and hMG-SF correspondingly.(D) ADP and ATP responses in PB-M(−). Note the absence of significant[Ca2+]i transients in response to ADP in macrophages. (E) Statisticalanalysis for the amplitudes of [Ca2+]i transients. Maximum amplitude of[Ca2+]i transient for each responsive cell is presented as a dot in thecorresponding category (***: p<0.001 by Student's t-test). (F)Percentages of ADP-responsive cells among all different cell typesanalyzed.

FIG. 5 A-D. Microglial progenitors. (A) Primitive hemagioblasts could beidentified as KDR+CD235a+ in the adherent fraction of day 6 cultures.(B) Representative plot of the sorting gate used to isolate CD14+CX3CR1+microglial progenitors via FACS between day 25 and 50 ofdifferentiation. (C) Phase contrast image of plated microglialprogenitors two days after isolation. Scale bar is 500 μm. (D) Graphshowing the performance of 2 ESC and 15 iPSC lines in generating CD14+myeloid progenitors, quantified by flow cytometry.

FIG. 6. Characterization of human primary microglia (hMG). Panel ofrepresentative images of hMG in phase contrast, and afterimmunofluorescent labeling for IBA1 and CD11c. Scale bars from left toright are 500 μm, 200 μm and 200 μm.

FIG. 7. Dendrogram showing hierarchical clustering of our RNA sequencingdata with data obtained from an independent study (Muffat et al., 2016)on iPSC-derived microglia. Samples termed, or including the terms, fMG,pMG and/or NPC correspond to those samples from Muffat et al., 2016.Analysis is based on global RNA expression after batch correction.

FIG. 8 A-D. Intracellular Ca2+ transients in human peripheral bloodmacrophages. Four example traces of intracellular Ca2+ changes during(A) ADP or (B) ATP application in macrophages polarized with (LPS,INFγ),(IL4,IL13) and (IL10). Bars represent duration of ADP (in A) or ATP (inB) application. Statistical analysis for the amplitudes of [Ca2+]itransients of (C) ATP-responsive or (D) ADP-responsive macrophages.Maximum amplitude of [Ca2+]i transient for each responsive cell ispresented as a dot.

FIG. 9. Schematic illustration of an exemplary protocol for generationof microglia from pluripotent stem cells, in which the “alternative” or“A” medium (described in Example 3) is used for the final microglialdifferentiation step.

FIG. 10. Results of immunofluorescence staining of microglial cellsdifferentiated from microglial precursors using the “alternative” or “A”medium described in Example 3. Staining for IBA1 and CD11c is shown.Nuclei are stained with DAPI. The left and right panels are images fromdifferent experiments where different lines and different seedingdensities of cells were used.

FIG. 11. Data showing that microglial cells differentiated frommicroglial precursors using the “alternative” or “A” medium described inExample 3 were able to phagocytose carboxylated latex beads.Representative flow cytometry histograms showing that microglial cellsdifferentiated from microglial precursors using the “alternative” or “A”medium described in Example 3 in two independent experiments (iPSC1 andiPSC2) were able to phagocytose YG-labeled carboxylated microspheres.Undifferentiated iPSCs were used as negative control (iPSCs-Negative).

FIG. 12. Heat-map of the released cytokine profiles of iPSC-MG culturedeither in the “A” or the “R” medium (as described in Example 3) ontoeither tissue-culture treated plastic (Plastic) or ultra-low attachmentplates (ULA), human primary microglia and peripheral blood-derivedmacrophages polarized to M(LPS, INFγ), M(IL4, IL13) and M(IL10) ornon-polarized M(−). Data show that the cytokine profiles of “A” or “R”microglia are very similar and cluster together with human primarymicroglia. All microglial cells create a distinct cluster that isseparate from macrophages.

DETAILED DESCRIPTION OF THE INVENTION

Some of the main aspects of the present invention are summarized abovein the Summary of the Invention section of this patent disclosure.Additional aspects are described in the Examples, Figures, and Claimssections of this disclosure. This Detailed Description of the Inventionprovides certain additional description and is intended to be read inconjunction with, and combined with the disclosure of, all othersections of this patent disclosure. Furthermore, the various embodimentsdescribed in each section of this disclosure can be combined in variousdifferent ways, and all such combinations are intended to fall withinthe scope of the invention. Headings and subheadings used anywhere inthis patent disclosure are provided for convenience and ease ofreference/reading only, and do not denote limitations of the variousaspects or embodiments of the invention described herein, which is to beunderstood by reference to the specification as a whole.

Definitions & Abbreviations

While certain terms are defined immediately below, each of these termsmay be more fully defined by their context of use and by reference tothe specification as a whole. Terms not specifically defined immediatelybelow may be defined elsewhere in this patent disclosure, of theirmeanings may be clear from the context in which the terms are used, orelse the terms are used in accordance with their usual meaning—ascommonly understood by one of ordinary skill in the art to which thisinvention pertains. For example, The Dictionary of Cell and MolecularBiology (5th ed. J. M. Lackie ed., 2013), the Oxford Dictionary ofBiochemistry and Molecular Biology (2d ed. R. Cammack et al. eds.,2008), and The Concise Dictionary of Biomedicine and Molecular Biology,P-S. Juo, (2d ed. 2002) can provide one of skill with generaldefinitions of some terms used herein.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents, unless the contextclearly dictates otherwise. The terms “a” (or “an”), as well as theterms “one or more,” and “at least one” can be used interchangeably.

Furthermore, “and/or” is to be taken as specific disclosure of each ofthe two specified features or components with or without the other.Thus, the term “and/or” as used in a phrase such as “A and/or B” isintended to include A and B, A or B, A (alone), and B (alone). Likewise,the term “and/or” as used in a phrase such as “A, B, and/or C” isintended to include A, B, and C; A, B, or C; A or B; A or C; B or C; Aand B; A and C; B and C; A (alone); B (alone); and C (alone).

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Wherever embodiments are described with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” areincluded.

Where a numeric term is preceded by “about” or “around” or“approximately,” the term includes the stated number and values ±10% ofthe stated number.

The term “pluripotent stem cells” or “PSCs” has its usual meaning in theart, i.e., self-replicating cells that have the ability to develop intoendoderm, ectoderm, and mesoderm cells. In some embodiments PSCs arehuman PSCs. PSCs include embryonic stem cells (ESCs) and inducedpluripotent stem cells (“iPS cells” or “iPSCs”). The terms ES cells andiPS cells have their usual meaning in the art.

As used herein the phrase “substantially pure” refers to a population ofcells wherein at least 95% of the cells have the recited phenotype. Inall embodiments that refer to a “substantially pure” cell population,alternative embodiments in which the cell populations have a lower orhigher level of purity are also contemplated. For example, in someembodiments, instead of a given cell population being “substantiallypure” the cell population may be one in which at least 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%of the cells, or 100% of the cells, have the recited phenotype.

By “subject” or “individual” or “patient” is meant any subject,particularly a mammalian subject, for whom diagnosis, prognosis, ortherapy is desired, or from whom a microglial cell is to be generatedusing a method as described herein. Mammalian subjects include humans,domestic animals, farm animals, sports animals, and zoo animalsincluding, e.g., humans, non-human primates, dogs, cats, guinea pigs,rabbits, rats, mice, horses, cattle, pigs, and so on. In someembodiments the subjects are human.

Terms such as “treating” or “treatment” or “to treat” refer totherapeutic measures that cure, slow down, lessen symptoms of, and/orhalt progression of a diagnosed pathologic disease or disorder. Thus,those in need of treatment include those already with the disorder. Incertain embodiments, a subject is successfully “treated” for a diseaseor disorder if the subject shows, e.g., total, partial, permanent, ortransient, alleviation or elimination of any symptom associated with thedisease or disorder.

“Prevent” or “prevention” refers to prophylactic or preventativemeasures that prevent and/or slow the development of a pathologicdisease or disorder. Thus, those in need of prevention include thoseprone to or susceptible to the disease or disorder. In certainembodiments, a disease or disorder is successfully prevented accordingto the methods provided herein if the subject develops, transiently orpermanently, fewer or less severe symptoms associated with the diseaseor disorder, or a later onset of symptoms associated with the disease ordisorder, than a subject who has not been subject to the methods of theinvention.

Additional Description

Various methods for the generation of microglial cells and microglialprogenitor cells are described in the Summary of the Invention,Examples, and Claims sections of this patent disclosure. The pluripotentstem cells used in such methods can be any suitable type of pluripotentstem cells. In one embodiment the pluripotent stem cells are ESCs oriPSCs—each of which is well known in the art. Where iPSCs are used, suchcells may have been “reprogrammed” to the pluripotent state from anon-pluripotent state using any suitable means known in the art,including, but not limited to, modified RNA-based methods, Sendai virusbased methods, and the like. Furthermore, such cells may have beenreprogrammed to the pluripotent state using any suitable cocktail ofreprogramming factors known in the art.

Unless a specific type of cell/tissue culture media is specified, anysuitable cell/tissue culture media known in the art can be used. Manysuch types of media are known and commercially available. In someembodiments, certain chemically-defined and/or serum free media typesare used.

In some embodiments, media suitable for use in maintaining pluripotentstem cells is used. In some such embodiments such a medium is mTeSR1medium from Stem Cell Technologies. However, one of skill in the artwill recognize that there are several other types of media that areequivalent to mTeSR medium in terms of their suitability for use inmaintaining pluripotent stem cells—any of which could be used. Typicallysuch media will contain one or more pluripotency factors to facilitatethe maintenance of cells in a pluripotent state. In certain embodimentsvariants of such media are used that do not comprise these pluripotencyfactors. For example, in certain embodiments where mTeSR1 medium isused, a variant of the mTeSR1 medium (sometimes referred to herein as“mTeSR1 Custom” medium) that does not comprise lithium chloride, GABA,pipecolic acid, bFGF or TGFβ1 is used. The composition of mTeSR1 mediumis known in the art and described in, for example, Ludwig et al., 2006(Nat Methods. 2006 August; 3(8):637-46; “Feeder-Independent Culture ofHuman Embryonic Stem Cells”), the contents of which are herebyincorporated by reference.

In some embodiments, media suitable for use in differentiatingpluripotent stem cells is used. Such media typical do not comprisepluripotency factors.

In some embodiments, media suitable for culturing hematopoietic cells isused. In some such embodiments such the medium is StemPro-34 fromThermoFisher. The composition of StemPro-34 medium is known in the artand described in, for example, EP 0891419 A4 entitled “HematopoieticCell Culture Nutrient Supplement” and WO1997033978A1, the contents ofwhich are hereby incorporated by reference. However, one of skill in theart will recognize that there are several other types of media that areequivalent to StemPro-34 medium in terms of their suitability for use inculturing hematopoietic cells—any of which could be used.

Many of the embodiments of the present invention involve certain factorsto be used in (or excluded from) the compositions and methods describedherein, for example as media supplements. These include, but are notlimited to, bFGF, SCF, VEGFA, IL-3, TPO, M-CSF, FLT3 ligand, GM-CSF,GABA, pipecolic acid, bFGF, TGFβ1, IL-34, GM-CSF, M-CSF, NGF-β and CCL2.Each of these factors is well known in the art, including the full namesof each of these factors in the cases where acronyms or otherabbreviations are used. Furthermore, all of these factors are availableto the public from multiple sources, including commercial sources.Exemplary amounts/concentrations for use of each of these factors in themethods and compositions of the present invention are provided in theExamples section of this patent disclosure. For all embodiments wherespecified amounts are referred to, amounts that are “about” thespecified amount are also intended. Furthermore, one of skill in the artwill recognize that in some situations further deviations from thespecified amounts can be used, and will be able to determine how much ofeach factor to use by performing routine testing, optimization,dose-response studies, and the like, for example to reduce or increasethe specified amounts, as long as the amounts used still achieve thestated effect—e.g. the stated differentiation effect. For example, insome embodiments specified amounts of the specified agents may bereduced to 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%,or 90% of the stated amounts. Similarly, in some embodiments thespecified amounts of the specified agents may be increased by 10%, by20%, by 30%, by 40%, by 50%, by 60%, by 70%, by 80%, by 90%, by 100%, by150%, by 200%, by 300%, by 400%, or by 500% of the stated amounts.Similarly, where specified factors are referred to, one of skill in theart will recognize that analogs, variants, or derivatives of suchfactors can also be used as long as the analogs, variants, orderivatives have the same general function/activity as the specifiedfactors.

Similarly, where exemplary times/timing for using of each of thesefactors, or indeed for performing any other step of any method providedherein, are specified, times/timing that is “about” the specified timeis also intended. Furthermore, one of skill in the art will recognizethat in some situations further deviations from the specifiedtimes/timing can be used, and will be able to determine how the time canbe adjusted by performing routine testing, optimization, dose-responsestudies, and the like, for example to reduce or increase the specifiedtimes/timing as long as the times/timing still achieves the statedeffect—e.g. the stated differentiation effect. For example, in someembodiments specified times may be reduced to 10%, or 20%, or 30%, or40%, or 50%, or 60%, or 70%, or 80%, or 90% of the stated times.Similarly, in some embodiments the specified times may be increased by10%, by 20%, by 30%, by 40%, by 50%, by 60%, by 70%, by 80%, by 90%, by100%, by 150%, by 200%, by 300%, by 400%, or by 500% of the statedtimes.

Unless otherwise stated herein, routine and well known methods andcompositions for cell culture are to be used in carrying out the methodsof the present invention, including routine methods for preparing media,exchanging media, spinning down supernatants to recover non-adherentcells, etc. In some embodiments the culture methods of the presentinvention used for the generation of microglia or microglial progenitorsfrom pluripotent stem cells employ monolayer cultures. In otherembodiments the culture methods of the present invention used for thegeneration of microglia or microglial progenitors from pluripotent stemcells do not employ the use of feeder cells/feeder layers. In furtherembodiments the culture methods of the present invention used for thegeneration of microglia or microglial progenitors from pluripotent stemcells do not employ, and/or do not require, embryoid body (EB)formation.

Many of the embodiments of the present invention describe cellpopulations in terms of their expression of certain cell markers. Thesemarkers include, but are not limited to, CD14, CX3CR1, CD11b, CD11c,P2RY12, IBA-1, and TMEM119. Each of these markers is well known in theart, including the full names of each of these markers—in the caseswhere acronyms or other abbreviations are used. When any of thesemarkers is referred to with a “+” symbol (e.g. marker+, e.g. CD14+), the“+” symbol means that the cell is positive for that marker—i.e.expresses detectable levels of that marker. One can determine if a cellexpresses a detectable level of any of these markers using standard androutine methods known in the art, including those involvingantibody-based detection, mRNA detection, and the like.

Several of the methods described herein involve isolating/separatingCD14+ and/or CX3CR1+ microglial progenitor cells from other cell types.In some embodiments these methods involve contacting the cells (e.g. amixed cell population) with an anti-CD14 antibody and/or a CX3CR1antibody. Then any suitable method known in the art can be used toseparate cells bound by the antibody from cells not bound by theantibody. One such method is fluorescence activated cell sorting (FACS).Other suitable methods include those that utilize magnetic beads. Suchmethods typically involve contacting with both the desired antibody andwith magnetic beads, wherein the magnetic beads are constructed suchthat they can directly or indirectly bind to the antibody. Then, amagnet can be used to separate cells bound by the antibody from cellsnot bound by the antibody. Any suitable anti-CD14 antibody oranti-CX3CR1 antibody know in the art can be used. Exemplary antibodiesare listed in the Examples section of this patent application.Furthermore, an exemplary commercially available kit for magnetic beadbased isolation of CD14+ cells is identified in the Examples section ofthis patent application.

In some embodiments the present invention provides tissue culture media,tissue culture media supplements, and various kits useful in performingthe various methods described herein.

In one embodiment, the present invention comprise a kit for generatingmicroglial progenitor cells from pluripotent stem cells, the kitcomprising any two or more factors selected from the group consistingof: BMP4, bFGF, SCF, VEGFA, SCF, IL-3, TPO, M-CSF,FLT3 ligand, M-CSF,and GM-CSF. In some embodiments the kit comprises each of these factors.

In one embodiment, the present invention comprise a kit for generatingmicroglial progenitor cells from pluripotent stem cells, the kitcomprising any two or more factors selected from the group consistingof: BMP4, bFGF, SCF, VEGFA, SCF, IL-3, TPO, M-CSF,FLT3 ligand, M-CSF,and GM-CSF, IL-34, NGF-β and CCL2. In some embodiments the kit compriseseach of these factors.

In some embodiments such kits comprise one or more media supplementcompositions, each present in a separate container, selected from thegroup consisting of: (a) a first media supplement/composition comprisingBMP4, (b) a second media supplement/composition comprising: one or moreof (e.g. each of) bFGF, SCF, and VEGFA, a third mediasupplement/composition comprising: one or more of (e.g. each of) SCF,IL-3, TPO, M-CSF, and FLT3 ligand, a fourth media supplement/compositioncomprising: one or more of (e.g. each of) M-CSF, FLT3 ligand, andGM-CSF, and a fifth media supplement/composition comprising: either (i)one or more of (e.g. each of) GM-CSF and IL-34 or (ii) one or more of(e.g. each of) M-CSF, GM-CSF, NGF-β and CCL2. Where the kit is to beused for preparation of microglial progenitors, each of the first tofourth media supplements/compositions may be included in the kit. Wherethe kit is to be used for preparation of microglial cells, all fivemedia supplements/compositions may be included in the kit. The kitsdescribed herein may also comprise tissue culture medium, such as amedium suitable for the maintenance or differentiation of pluripotentstem cells, and/or a hematopoietic cell medium. In some embodiments themedium does not comprise pluripotency factors.

The kits described herein may also include an anti-CD14 antibody, ananti-CX3CR1 antibody, or both an anti-CD14 antibody and an anti-CX3CR1antibody.

The kits may optionally comprise instructions for use, one or morecontainers, one or more antibodies, or any combination thereof. A labeltypically accompanies the kit, and includes any writing or recordedmaterial, which may be electronic or computer readable form (e.g., disk,optical disc, memory chip, or tape) providing instructions or otherinformation for use of the kit contents.

In some embodiments the present invention also provides tissue culturemedia, which may optionally be provided with certain media supplements.For example, in one embodiment the present invention provides a mediumsuitable for use in maintaining pluripotent stem cells, wherein themedium does not comprise pluripotency factors. For example, in some suchembodiments the medium does not comprise lithium chloride, GABA,pipecolic acid, bFGF or TGFβ1. Optionally, such media may comprise, orbe provided with, BMP4 as a media supplement. In another embodiment thepresent invention provides a medium suitable for culture ofhematopoietic cells. Optionally, such media may comprise, or be providedwith, a media supplement/composition comprising: one or more of (or eachof) bFGF, SCF, and VEGFA; and/or a media supplement/compositioncomprising: one or more of (or each of) SCF, IL-3, TPO, M-CSF, and FLT3ligand; and/or a media supplement/composition comprising: one or more of(or each of) M-CSF, FLT3 ligand, and GM-CSF. In some such embodimentsthe media is serum free media. In some such embodiments the media is achemically-defined media.

The practice of the present invention will employ, unless otherwiseindicated conventional techniques of cell biology, molecular biology,cell culture, immunology and the like which are in the skill of one inthe art. These techniques are fully disclosed in the current literatureand reference is made specifically to Sambrook, Fritsch and Maniatiseds., “Molecular Cloning A Laboratory Manual, 2nd Ed., Cold SpringsHarbor Laboratory Press, 1989); the series Methods of Enzymology(Academic Press, Inc.); and Antibodies: A Laboratory Manual, Harlow etal., eds., (1987).

EXAMPLES Example 1—Generation of Microglia from Human Pluripotent StemCells

As microglia cells arise from myeloid progenitors in the yolk sackduring embryonic development, we sought to establish a serum- andfeeder-free protocol to differentiate human PSCs towards the myeloidlineage. The protocol that we generated is illustrated in schematic formin FIG. 1A). Building upon previous studies (Yanagimachi et al., 2013),we induced primitive streak-like cells through BMP4 signaling, obtaininga KDR+CD235a+ population of primitive hemangioblasts (Sturgeon et al.,2014) (Figure S1A). CD45+CX3CR1− microglial progenitors appeared in thesupernatant fraction of the culture by day 16, while CX3CR1 wasupregulated between day 20 and 25. In contrast, the adherent populationcontained only a small fraction of CD45+CX3CR1+ progenitors.Interestingly, a subset of the CD45+CX3CR1-population upregulated CD14around day 16, before the upregulation of CX3CR1 (FIG. 1B). Between day25 and day 50, 82±5% of the CD14+ cells co-expressed CX3CR1. Theprotocol's efficiency to generate microglial progenitors, based on CD14expression, was 68±4% across seventeen lines tested (FIG. 5D), whichincluded two ESC and fifteen iPSC lines. Microglial progenitors werecontinuously generated in the supernatant fraction for up to one monthand they were isolated once per week with an average yield of 224±42×103cells per isolation, for every 100×103 PSCs plated. Microglialprogenitors were isolated either via FACS sorting (FIG. 5 B, C) ormagnetic bead-based separation for further differentiation or forlong-term storage in liquid nitrogen. Thawed progenitors retained theirdifferentiation capacity, with a post-thaw viability of 57±5%.

Generation of Microglia

The protocol that we developed for generation of microglia isillustrated in schematic form in FIG. 1A). Microglial progenitors wereproduced from iPSCs as described in Example 1. IL-34 and GM-CSFstimulation for two weeks induced the differentiation of isolatedmicroglial progenitors into microglia. The iPSC-derived microglia cells(iPSC-MG) grew as adherent cells, extended many processes and exhibitedmorphology typical of microglial cells (FIG. 2A). Furthermore,time-lapse video microscopy of iPSC-MG showed their processes to behighly motile, constantly scanning the microenvironment, similarly tomicroglia cells in vivo (Davalos et al., 2005; Nimmerjahn, 2012).iPSC-MG expressed known antigenic markers including IBA-1, CD11c,TMEM119, P2RY12, CD11b and CX3CR1 (FIG. 2A-C). Of note, Thermanoxplastic coverslips seemed to provide the optimal surface environment ascells appeared more ramified. Commercially available human primarymicroglia cells (hMG) were used for comparison (FIG. 6).

iPSC-Derived Microglia Resemble Human Primary Microglia

To further confirm the identity of iPSC-MG, whole transcriptome analysiswas performed with next generation deep RNA sequencing (RNAseq). iPSC-MGfrom six unrelated healthy donors were compared to peripheralblood-derived macrophages (PB-M(−)), macrophages polarized toM(LPS,IFNγ), M(IL4,IL13) and M(IL10), primary human hepatic macrophages(hhM) and primary human microglia cultured in serum-containing mediasupplied by the provider (hMG) or in our serum-free media (hMG-SF). Weobtained high quality sequencing reads (mean Phred quality score >38.4)of which more than 86.2% mapped to human genome hg19 by the STARaligner. A total of 18,516 genes were considered expressed and used forfurther analysis.

iPSC-MG, hMG and hMG-SF clustered together in a hierarchical clusteranalysis using all the expressed genes showing high degree of similarityat the global gene expression level (Spearman's correlation coefficient0.901˜0.997) and were distinct from all macrophage subtypes (FIG. 3A).We then performed hierarchical cluster analysis including data obtainedfrom an independent study (Zhang et al., 2016) that isolated primaryCD45+ cells from human brain extracts (termed “Myeloid” in FIG. 3B). TheiPSC-MG samples clustered together with the “Myeloid” samples, whilePB-M created a distinct cluster and hhM clustered separately, appearingthe most dissimilar to any of the tested samples.

Three recent studies (Bennett et al., 2016; Butovsky et al., 2014;Hickman et al., 2013) have provided datasets with unique genes expressedin microglia from primary rodent cells. We next selected genes that wereidentified in at least two of these studies and assessed theirexpression in our samples (Table 1).

TABLE 1 iPSC- iPSC- iPSC- iPSC- hMG- MG/ MG/ MG/ MG SF hhM PB-M(−)hMG-SF hhM PB-M(−) Common in all TREM2 10.0 8.7 4.5 8.6 1.2 2.2 1.2 3studies SLCO2B1 9.7 9.7 7.1 6.4 1.0 1.4 1.5 HEXB 9.6 9.1 8.5 9.3 1.0 1.11.0 GPR34 8.8 7.6 3.4 4.3 1.2 2.5 2.0 OLFML3 3.4 5.7 6.9 1.6 0.6 0.5 2.2SLC2A5 2.9 4.3 3.8 6.9 0.7 0.8 0.4 P2RY12 2.4 1.2 −3.6 −2.9 2.0 −0.7−0.8 P2RY13 1.1 3.0 −1.6 −1.1 0.4 −0.7 −1.0 TMEM119 −0.5 2.7 4.7 −2.5−0.2 −0.1 0.2 LIPH −4.4 −3.4 −1.7 −2.2 1.3 2.6 2.0 CTSD 11.0 12.5 9.311.7 0.9 1.2 0.9 TGFBR1 9.2 7.4 9.7 7.6 1.2 1.0 1.2 ENTPD1 8.3 6.3 5.16.0 1.3 1.6 1.4 IL10RA 8.2 7.0 7.6 8.5 1.2 1.1 1.0 LAIR1 7.2 8.4 7.2 8.00.9 1.0 0.9 Butovsky & CYSLTR1 5.6 1.4 0.7 1.7 3.8 7.6 3.3 Bennett BLNK4.8 4.8 0.5 2.5 1.0 9.7 2.0 RAB3IL1 4.8 4.7 2.8 3.0 1.0 1.7 1.6 GOLM14.5 3.8 6.8 3.5 1.2 0.7 1.3 PMEPA1 4.1 3.6 4.2 2.2 1.1 1.0 1.9 OPHN1 3.63.4 2.6 3.5 1.0 1.4 1.0 CCR5 3.6 6.1 4.7 5.5 0.6 0.8 0.6 F11R 8.0 7.66.4 8.6 1.1 1.3 0.9 ADORA3 7.0 7.4 1.4 1.7 0.9 4.9 4.1 SPINT1 6.9 5.23.0 7.8 1.3 2.3 0.9 Butovsky & CCL4 5.0 6.5 8.0 1.7 0.8 0.6 2.9 HickmanCRYBB1 3.2 3.2 1.0 −1.1 1.0 3.2 −2.9 CX3CR1 0.8 −5.7 −1.2 −3.1 −0.1 −0.6−0.2 ANG 0.4 0.6 0.6 1.5 0.7 0.8 0.3 LAG3 −2.4 0.2 −2.0 −1.6 −11.1 1.21.4 Hickman & GAL3ST4 3.0 4.3 2.7 −0.8 0.7 1.1 −3.9 Bennett

Of the 31 selected genes, 29 were expressed (defined as at least 1 CPM)by hMG and 28 by iPSC-MG. Overall, the expression levels of these geneswere comparable with the exception of P2RY13, which was 2.6 times lessin iPSC-MG and CYSLTR1, which was 3.8 times less in hMG. Moreover, LIPHhad very low expression (<1 CPM) in all human cell types tested, whilehMG showed low expression of CX3CR1, and iPSC-MG low expression ofTMEM119 and LAG3. However, CX3CR1 and TMEM119 proteins were detected byFACS and immunofluorescent staining respectively.

Comparisons with tissue-resident hepatic macrophages and peripheralblood-derived macrophages highlighted 11 genes (TREM2, SLCO2B1, GPR34,P2RY12, P2RY13, ENTPD1, BLNK, RAB3IL1, ADORA3, CRYBB1, GAL3ST4) thatwere consistently higher in both iPSC-derived and primary microglia(Table 1).

The study from Butovsky et al., proposed six genes, namely C1QA, GAS6,GPR34, MERTK, P2RY12 and PROS1 as unique signature in fetal and adulthuman primary microglia. Indeed, their high expression was consistentamong all our microglial samples (iPSC-MG, hMG, hMG-SF) as well as inmicroglial samples from two independent studies (Muffat et al., 2016;Zhang et al., 2016) (FIG. 3C), whereas macrophages and neural progenitorcells (NPCs) showed lower levels (t-test: P<0.05 between microglia andmacrophages or NPCs in all 6 genes). Table 2, which relates to FIG. 3C,shows the expression of the six human microglial validated signaturegenes in the microglial samples as determined by RNAseq. The values inTable 2 are log 2 transformed CPM.

TABLE 2 iPSC- iPSC- iPSC- iPSC- iPSC- iPSC- hMG- MG1 MG2 MG3 MG4 MG5 MG6SF hMG C1QA 9.2 8.5 9.1 9.5 7.9 7.9 8.6 10.2 GAS6 7.0 6.0 7.6 7.0 6.76.2 5.4 5.0 GPR34 9.2 8.5 9.1 8.9 8.2 8.0 7.6 8.5 MERTK 7.6 7.3 7.7 8.58.8 8.6 8.6 8.3 P2RY12 4.2 0.4 3.0 3.2 0.9 1.6 1.2 5.7 PROS1 6.2 6.4 6.86.8 6.0 5.7 4.7 4.5

The cytokine profiles of human iPSC-derived and primary microglia werefound to be similar, but to differ from those of peripheral bloodderived macrophages. We analyzed proteins released by iPSC-MG, hMG,hMG-SF, and PB-M, including differentially polarized macrophages (FIG.3D). Interestingly, similarities between iPSC-MG and primary microgliadrastically increased when we replaced their culturing medium(containing serum, hMG) with our differentiation medium (hMG-SF), as thePearson's correlation coefficient increased from R=0.473 to R=0.824. Ofnote, hMG showed upregulation of cytokines, such as RANTES, I-TAC, BAFF,GR0-a and MIP3a, which are typically released upon inflammation, and infact, were also expressed by M(LPS,IFNγ) macrophages (arrows in FIG.3D). Nevertheless, microglia samples clustered together and away fromPB-M polarized as M(−), M(LPS,IFNγ), M(IL4,IL13) and M(IL10).

Our iPSC-MGs were also found to be functional phagocytes. iPSC-MG, hMG,hMG-SF, PB-M and undifferentiated iPSCs were challenged with a givenamount of fluorescently labeled carboxylated latex microspheres percell. Flow cytometry analysis showed that the majority of iPSC-MG wereable to phagocytose (90±6%). These results were comparable to both hMGand hMG-SF. As expected, M(−) macrophages were also able to engulfmicrospheres while undifferentiated iPSCs were not (FIG. 3E).

Our iPSC-MGs were also found to release intracellular Ca2+ in responseto ADP. The microglial signature gene P2RY12 encodes a Giprotein-coupled purinergic receptor (Haynes et al., 2006) that respondsto ADP resulting in intracellular Ca2+ ([Ca2+]i) transients, whereasPB-M do not express the receptor and consequently do not respond to ADP(Moore et al., 2015). Thus, ADP-induced [Ca2+]i transients are used todifferentiate between microglia and macrophages. When we stimulatediPSC-MG, hMG, hMG-SF and PB-M with 50 μM ADP for 60 seconds, onlymicroglial cells responded to ADP (FIG. 4A-C). The peak amplitude of ADPresponses in iPSC-MG (FIG. 4E) as well as the number of responsive cells(FIG. 4F) were higher than either hMG or hMG-SF. None of thedifferentially polarized PB-M respond to ADP, but [Ca2+]i transientswere reliably observed upon stimulation with 100 μM ATP (FIG. 4D andFIG. 8).

Summary & Discussion

In vitro hematopoietic differentiation of PSCs is equivalent to the invivo primitive hematopoiesis rather than definitive hematopoiesis. Thismay explain why PSC-derived hematopoietic progenitors fail to producelong-term multi-lineage reconstitution (Vanhee et al., 2015). Wehypothesized that PSC-derived myeloid progenitors might resembleprimitive yolk sac myeloid progenitors, and therefore might give rise tomicroglia in vitro—as occurs during embryonic development. Bystimulating PSCs with a myeloid inductive medium, we produced aKDR+CD235a+ primitive hemangioblast population and recapitulated theprogression from CD45+CX3CR1− to CD45+CX3CR1+ microglial progenitors invitro.

To ensure robustness and reproducibility of the protocol we tested apanel of 17 PSC lines, including iPSCs from MS, AD, PD patients orhealthy individuals generated using different reprogramming strategies(e.g. mRNA/miRNA, Sendai virus). Fibroblasts were obtained from bothmale and female donors with ages ranging from 25 to 68. We were able toobtain microglial progenitors from all lines, with an average yield of2-3 progenitors per undifferentiated PSC. As expected from such adiverse panel, the yield of progenitors varied across the lines, with noapparent correlation to a specific disease, reprogramming method or sexand age of the donor.

Our microglial progenitors gave rise to microglia that expressed typicalmarkers, were ramified with highly motile processes capable of scanningthe microenvironment, and were are able to phagocytose with anefficiency equivalent to that of normal human microglia.

While a definition of human microglial identity is not well established,recent genome-wide studies in mouse have provided datasets to facilitatethe distinction of microglia from other myeloid or CNS cell types(Bennett et al., 2016; Butovsky et al., 2014; Hickman et al., 2013).Therefore, we compared the global mRNA expression of iPSC-MG to that ofprimary microglia and both peripheral blood-derived and hepaticmacrophages to evaluate the proposed “signature genes” in humanmicroglia. We included samples with different genetic backgrounds. Ouranalyses clearly showed that iPSC-MG clustered away from bothcirculating and other tissue-specific macrophages and clustered togetherwith primary microglia cells and CD45+ cells (called “myeloid”),isolated from human brain extracts (Zhang et al., 2016). Furthermore ouriPSC-MG expressed six genes suggested as being unique to human microglia(Butovsky et al., 2014), in addition to many other genes that areenriched in mouse microglia (Table 1).

The cytokines released by iPSC-MG were found to have similar profiles tothose of hMG but distinct from those of PB-M—independent of theirpolarization status. Of note, hMG clustered more tightly with iPSC-MGwhen cultured in our medium (hMG-SF). Without wishing to be bound bytheory, we hypothesize that the reason is the presence of serum in thehMG culture medium. In vivo, microglia reside “behind” the blood-brainbarrier, and the presence of serum components triggers their activation(Ransohoff and Perry, 2009). Indeed, hMG cultured in serum showedincreased levels of inflammatory molecules such as RANTES, GR0-A, I-TAC,BAFF and MIP3a, similarly to M(LPS,IFNγ) pro-inflammatory macrophages.

We also showed that iPSC-MG express a functional P2RY12 receptor at bothtranscript and protein levels. This receptor distinguishes rodent andhuman microglia from other myeloid cells (Butovsky et al., 2014) and itsactivation via ADP results in [Ca2+]i transients (Moore et al., 2015).All microglia cells (iPSC-MG, hMG and hMG-SF) showed ADP-evoked [Ca2+]itransients, while PB-M were unresponsive to ADP but showed [Ca2+]itransients upon exposure to ATP, indicating that they were healthy andfunctional.

While protocols to differentiate mouse ESCs into microglia are known(see, for example, Beutner et al., 2010; Napoli et al., 2009; Tsuchiyaet al., 2005) we found that such protocols are not effective with humaniPSCs (data not shown)—underscoring the difference between the rodentand human systems. Human microglia-like cells have previously beentrans-differentiated from peripheral blood (PB) monocytes, albeitwithout an extensive characterization of the microglia signature genesand functional studies (Etemad et al., 2012; Ohgidani et al., 2014).Muffat et al. (September 2016) described a human microglialdifferentiation protocol. We used RNAseq to compare microglia generatedby our methods to microglia generated using the methods of Muffat et al.This comparative data is provided herein (FIG. 3C and FIG. 7). Bothapproaches mimic embryonic development of microglia, which are derivedthrough a yolk sac progenitor defined as CD235a+. Both protocols useIL34 as the main driver to microglial lineage commitment and maturationin chemically defined media, and they both generate microglia withmotile processes that express typical makers and are able to performphagocytosis. Our strategy, based on monolayer cultures instead ofembryoid bodies (EBs) formation, is comparable in efficiency butrequires a smaller number of starting PSCs. Importantly, our protocoldoes not require the manual selection of specific EB morphology. Weisolate microglial progenitors via FACS or magnetic beads, enablinghigh-throughput applications such as compound and genetic screens.

In summary, the new protocols described herein provide iPSC-MG as a newsource of human microglia cells, which will complement studies in mousemodels to better understand the role of microglia in health and disease.The protocols described herein are highly reproducible, whetherperformed with lines from healthy subjects or diseased patients, andthus provide a valuable tool to investigate pathogenic mechanisms ofmicroglia dysfunction in neurological disorders. Furthermore, theinclusion of microglia in co-culture or three-dimensional systems, forexample those involving neurons and other glial cell types, shouldfacilitate in vitro disease-modeling to better recapitulate thecomplexity of the in vivo environment.

Example 2—Experimental Procedures

This Example provides details regarding the materials and methods usedto perform the studies outlined in Example 1.

Pluripotent Stem Cell Lines

Two human ESC lines (RUES1 and H9, both NIH approved) and 15 iPSC lineswere used in this study; these lines are described in the SupplementalInformation. One iPSC line was a gift from Dr. Ricardo Feldman and allother iPSC lines were reprogrammed at the NYSCF Research Institute.Human primary microglia and hepatic macrophages were purchased fromScienCell Research Laboratories.

Microglial Differentiation Protocol

PSCs differentiation was induced with mTeSR Custom medium (StemCellTechnologies), containing 80 ng/ml BMP4. At day 4 cells were inducedwith 25 ng/ml bFGF, 100 ng/ml SCF and 80 ng/ml VEGF in StemPro-34 SFM(with 2 mM GutaMAX, Life Technologies). Two days later, the medium wassupplemented with 50 ng/ml SCF, 50 ng/ml IL-3, 5 ng/ml TPO, 50 ng/mlM-CSF and 50 ng/ml Flt3 and from day 14 with 50 ng/ml M-CSF, 50 ng/mlFlt3 and 25 ng/ml GM-CSF. Between days 25-50 CD14+ or CD14+CX3CR1+progenitors were isolated and re-plated onto tissue culture-treateddishes or Thermanox plastic coverslips (all from Thermo Scientific) inMicroglial Medium (RPMI-1640, Life Technologies, with 2 mM GlutaMAX-I,10 ng/ml GM-CSF and 100 ng/ml IL-34). Medium was replenished every 3 to4 days for at least 2 weeks.

Tissue Culture & Cell Lines

All incubations were performed in a 37° C. incubator with 5% CO2 and allgrowth factors are human recombinant proteins purchased from R&DSystems, unless otherwise stated. All media contain 1×Penicillin/Streptomycin (P/S) or 1× Antibiotic-Antimycotic (LifeTechnologies). RT: room temperature.

RUES1 and H9 are NIH approved human ESC lines. All iPSC lines werederived from skin biopsies of de-identified donors upon specificinstitutional review board approvals and informed consent. The controliPSC lines 050643-01-MR-28495 (25 y.o. male), 050652-01-MR-28650 (45y.o. female), 050598-01-MR-49026 (53 y.o. male), 050598-01-MR-025 (53y.o. male), 051024-01-MR-005 (29 y.o. male), 050689-01-MR-012 (27 y.o.male), 050675-01-MR-012 (34 y.o. male), 050642-01-MR-001 (69 y.o. male),the PD line 050412-01-MR-49025 (63 y.o. male) and the AD line050948-01-MR-028 (40 y.o. female) were reprogrammed using New York StemCell Foundation's high-throughput automated system (see U.S. PatentApplication Publication No. 2013/0345094 and Paull et al., 2015) withthe mRNA/miRNA method (StemGent) (Paull et al., 2015). iPSC102 (56 y.o.male) was reprogrammed manually from a multiple sclerosis patient, usingmRNA/miRNA (Douvaras et al., 2014). 10001.198.01SV4Mut and10001.198.01SV5Mut are two distinct clones reprogrammed with Sendaivirus from a PD patient (68 y.o. male) with a GBA N370S mutation(Woodard et al., 2014) whereas 10001.198.01SV5WT is a CRISPR/Cas9corrected line. MJ2136 was a kind gift from Dr. Ricardo Feldman and wasreprogrammed with Sendai virus from a healthy control (Panicker et al.,2014).

The human primary microglia and hepatic macrophages were purchased fromScienCell along with Microglia Medium and Macrophage Medium (ScienCell).

PSC lines were cultured and expanded onto Matrigel-coated dishes inmTeSR1 medium (StemCell Technologies). Lines were passaged every 3-4days using enzymatic detachment with Stempro Accutase (LifeTechnologies) for 5 min and re-plated in mTeSR1 medium with 10 μM RockInhibitor (Y2732, Stemgent) for 24 hours.

Isolation of Myeloid Progenitors

Cells from the supernatant fraction of the cultures were incubated withCX3CR1 and/or CD14 conjugated primary antibodies (see Table 3) or theirrespective isotype controls for 40 min on ice. Cells were then washed inFACS buffer (PBS, 0.5% BSA, 2 mM EDTA, 20 mM Glucose), pelleted at 300 gfor 6 min and re-suspended in FACS buffer containing DAPI for dead cellexclusion. CD14+ or CD14+CX3CR1+ cells were isolated via FACS on anARIA-IIu™ Cell Sorter (BD Biosciences) using the 100 μm ceramic nozzle,and 20 psi.

Freezing and Thawing of the Myeloid Progenitors

Myeloid progenitor cells were frozen after isolation in cryogenic vials(Thermo Scientific) in freezing medium consisting of 90% FBS (LifeTechnologies) and 10% DMSO (Sigma-Aldrich). Cells were then transferredinto a Mr. Frosty (Thermo Scientific) container and placed overnight at−80° C. The next day, cryogenic vials were transferred to liquidnitrogen for long-term storage.

To thaw the cells, the cryogenic vial was transferred in a 37° C. waterbath for 1-2 min, until partially thawed. Under a laminar flow hood,RPMI-1640 medium was added to 5× the original volume of the vial. Cellwere then centrifuged at 300 g for 6 min, resuspended in the appropriateamount of medium and plated onto tissue culture treated plastic.

Detailed Microglial Differentiation Protocol

PSCs were plated onto Matrigel (BD Biosciences) in a 15×103 cells/cm2density in mTeSR1 medium containing 10 μM Rock Inhibitor for 24 hours.When individual colonies were visible (usually 2-4 days after plating),differentiation was induced by providing mTeSR Custom medium (StemCellTechnologies), containing 80 ng/ml BMP4. mTeSR Custom medium is mTeSR1medium without Lithium Chloride, GABA, Pipecolic Acid, bFGF and TGFβ1(Stem Cell Technologies). The medium was changed daily for 4 days, whencells were induced with StemPro-34 SFM (containing 2 mM GutaMAX-I, LifeTechnologies) supplemented with 25 ng/ml bFGF, 100 ng/ml SCF and 80ng/ml VEGF. Two days later, the medium was switched to StemPro-34containing 50 ng/ml SCF, 50 ng/ml IL-3, 5 ng/ml TPO, 50 ng/ml M-CSF and50 ng/ml Flt3. On day 10, the supernatant fraction of the cultures waspelleted, resuspended in fresh medium (same as before) and returned totheir dishes. On day 14, floating cells were pelleted, resuspended inStemPro-34 containing 50 ng/ml M-CSF, 50 ng/ml Flt3 and 25 ng/ml GM-CSFand replated back to their dishes. The procedure was repeated every fourdays. From day 24-52, a small amount of floating cells was processed forflow cytometry analysis to determine the peak of the CD14/CX3CR1 doublepositive progenitors. After the isolation of CD14+ or CD14+CX3CR1+progenitors, cells were plated onto tissue culture-treated dishes orThermanox plastic coverslips (all from Thermo Scientific) in a 40-50×103cells/cm2 in SF-Microglial Medium (RPMI-1640 from Life Technologiessupplemented with 2 mM GlutaMAX-I, 10 ng/ml GM-CSF and 100 ng/ml IL-34).Medium was replenished every 3 to 4 days for at least 2 weeks.

Peripheral Blood Derived Macrophages and Polarization

Macrophages were differentiated from isolated human mononuclear cellsobtained from peripheral blood of healthy individuals at the New YorkBlood Center as previously described (Pallotta et al., 2015). Briefly,CD14+ cells were isolated after Ficoll gradient and magnetic beads basedseparation using the EasySep Human CD14 Positive Selection Kit (STEMCELLTechnologies). Cells were then seeded in ultra-low attachment plates for5 days in a 5×105 cells/ml density and differentiated to macrophagesusing RPMI-1640 supplemented with 2 mM GlutaMAX-I, 10% heat-inactivatedhuman serum (Sigma-Aldrich) and 20 ng/ml M-CSF (PeproTech). Forpolarization, macrophages were kept in the same medium (M(−)), ortreated with 100 ng/ml LPS (Sigma-Aldrich) and 100 ng/ml IFNγ (M(LPS,IFNγ)), 40 ng/ml IL-4 and 20 ng/ml IL-13 (M(IL4,IL13)) or 40 ng/ml IL-10(M(IL10); all from PeproTech) macrophages.

Immunofluorescent Staining

Cells were washed 3× in PBS-T (PBS containing 0.1% Triton-X100) for 10min, incubated for 2 hours in blocking serum (PBS-T with 5% donkeyserum) and primary antibodies (see Table 3) were applied overnight at 4°C. The next day, cells were washed 3× in PBS-T for 15 min, incubatedwith secondary antibodies for 2 hours at room-temperature (RT), washed3× for 10 min in PBS-T, counterstained with DAPI for 15 min at RT andwashed 2× in PBS. Secondary antibodies were used at 1:500 dilution.Images were acquired using an Olympus IX71 inverted microscope, equippedwith Olympus DP30BW black and white digital camera. Fluorescent colorswere digitally applied using the Olympus software DP Manager or imageJ.

Flow Cytometry Analysis

Cells were enzymatically harvested by Accutase treatment for 5 min at37° C. and then scrapped with a cell lifter (Sigma-Aldrich). Cells werethen re-suspended in 100 μl of their respective medium containing theappropriate amount of fluorescence-conjugated antibodies (see Table 3)and were incubated on ice for 40 min shielded from light. Isotypecontrols or secondary antibodies only were used to measure the baselinebackground signal. DAPI or Sytox Green (Thermofisher) were used for deadcell exclusion. Analyses were performed on a five-laser BD BiosciencesARIA-IIu™ Cell Sorter or on a four-laser Attune NxT Flow Cytometer(ThermoFisher). Data were analyzed using BD FACSDiva™ software or FlowJoversion 9.9.4 (FlowJo LLC).

TABLE 3 Antibodies used for flow cytometry and immunofluorescentanalyses Name Host Vendor IBA1 Rabbit Wako P2RY12 Rabbit Alomone LabsCD11b-Alexa700 Mouse BD Pharmingen CD11c-PE Mouse BD PharmingenCX3CR1-PE Mouse R&D Systems CD14-APC Mouse BioRad CD45-v450 Mouse BDHorizon CD309-APC Mouse Miltenyi Biotec CD235a-PE Mouse BD Pharmingenanti-rabbit IgG-Alexa488 Donkey Life Technologies anti-mouseIgG-Alexa555 Donkey Life Technologies

Phagocytosis Assay

Phagocytosis assay was performed as previously described (Enomoto etal., 2013). Briefly, Fluoresbrite YG Carboxylate Microspheres 1.00 μm(Polysciences) were added to the dishes containing adherent microglialcells in a 200 microspheres/cell ratio. After incubating the cultures at37° C. for 3 hours, fluorescent images were acquired with an OlympusIX71 inverted microscope, equipped with Olympus DP30BW black and whitedigital camera. Then cells were washed 3× with PBS, treated withAccutase for 5 min and completely detached using a cell lifter. Aftercentrifuging, cells were resuspended in FACS buffer containing DAPI andanalyzed on a BD Biosciences ARIA-IIu™ Cell Sorter.

Cytokine Profiler and Clustering

For analysis of the secreted cytokine profile of microglial cells, theHuman XL Cytokine Array Kit of the Proteome Profiler Antibody Arrays(R&D Systems) was used according to the manufacturer's instructions.Supernatant was collected from the cultures and stored at −80° C. for upto 3 months. Membranes were directly visualized in a Kodak Image Station4000MM PRO and images were acquired using the Carestream MolecularImaging Software.

For analysis of the signals, images were imported to Image J and theProtein Array Analyzer plugin was used. The intensity reading of the twoidentical spots was then averaged and the mean value of 8 negativecontrols was subtracted from every value. Finally, data were expressedas intensity ratio compared to the mean intensity of the 6 referencespots (positive controls). Heatmap and clustering for the proteinprofiler analysis was generated using heatmap.2 in R version 3.3.1.

Intracellular Ca2+ Imaging

Cells were cultured onto Thermanox plastic coverslips (ThermoFisher) andwere incubated for 30 min at 37° C. with medium supplemented with thefluorescent Ca2+ dye Fluo-4/AM (2□M) mixed at 1:1 with Pluronic-127reagent (both from Invitrogen). Cells were subsequently washed twicewith RPMI-1640 media containing 1% BSA (Life Technologies), 1×GlutaMAX-I and 10 mM HEPES (Sigma-Aldrich) pH adjusted to 7.5. Cellswere allowed to recover for an additional 30 min to ensure dyeesterification. Coverslips were then transferred to a recording chambermounted onto an upright Olympus BX61 microscope. Fluorescence wasrecorded at 2 Hz by a cooled CCD camera (Hamamatsu Orca R2). Drugapplication was done via whole chamber perfusion at room temperature fora period of 60 s. [Ca²⁺]i transients are expressed in the form ofΔF(t)/F0, where F0 is a baseline fluorescence of a given region ofinterest and ΔF is the difference between current level of fluorescenceF(t) and F0. Fluctuations of ΔF(t)/F0 that were less than 0.05 wereconsidered as non-responses.

RNA Sequencing and Analyses

RNA isolation was performed using the RNeasy Plus Mini Kit (Qiagen) withQIAshredder (Qiagen). Cells were enzymatically detached after treatmentwith Accutase for 5 min and using a cell lifter. After centrifuging,cells were washed with PBS and resuspended in lysis buffer. Samples werethen stored at −80° C. until processed further according tomanufacturer's instructions. RNA was eluted in 30 μl RNase free ddH2Oand quantified with a NanoDrop 8000 spectrophotometer (ThermoScientific).

Single-ended RNAseq data were generated with the Illumina HiSeq 2500platform following the Illumina protocol. The raw sequencing reads werealigned to human hg19 genome using star aligner (version 2.5.0b).Following read alignment, featureCounts (Liao et al., 2014) was used toquantify the gene expression at the gene level based on Ensembl genemodel GRCh37.70. Genes with at least 1 count per million (CPM) in atleast one sample were considered expressed and hence retained forfurther analysis, otherwise removed. The gene level read counts datawere normalized using trimmed mean of M-values normalization (TMM)method (Robinson et al., 2010) to adjust for sequencing library sizedifference. Hierarchical cluster analysis based on transcriptome-widegene expression was performed using R programming language.

For re-analysis of microglia RNAseq data from Zhang et al. (Zhang etal., 2016), we downloaded the raw RNAseq data of “myeloid” cells fromgene expression omnibus (GEO: accession GSE73721). The RNAseq read datawas processed using the same star/featureCounts pipeline as describedabove and then the gene level read counts were combined with the genecount data of our samples. The merged data were normalized with the TMMapproach and then corrected for batch using linear regression.Hierarchical cluster analysis was used to illustrate the sampledissimilarity.

Similarly, for comparison with a recently published human iPSC-derivedmicroglial dataset from Muffat et al., 2016, we downloaded their RNAseqread data from GEO (accession GSE85839) and then applied the same RNAseqanalysis pipeline to obtain gene level count data which was merged withthe read count of the present samples. The merged data was normalizedand batch corrected before carrying out hierarchical cluster analysis.

Statistics

Frequencies were calculated in excel and expressed as Mean±StandardError of the Mean (SEM). Statistical analysis of [Ca2+]i transientsamplitude was performed using unpaired Student's t-test to compare meanvalues in excel. The P value significance of the cluster partition forthe dendrogram based on the six signature genes was estimated as thefraction of 1000 repeated permutations (shuffling gene expression valueswithin each gene) in which the cluster center distance obtained fromk-means cluster with two centers was more extreme than that in theoriginal data. Pearson's correlation coefficient between iPSC-MG and hMGor hMG-SF for cytokine release data was calculated using GraphPad Prism6.

Example 3

Alternative Protocols for the Generation of Microglia from PluripotentStem Cells

This example provides alternative exemplary protocols for the generationof microglia from pluripotent stem cells. In these protocols,CD14+/CX3CR1+ microglial progenitors are isolated via FACS, or based ononly CD14 expression using magnetic bead-based isolation, and theisolated CD14+ progenitors are then plated onto tissue culture treatedplastic in either an alternative (“A”) microglial differentiation medium(comprising M-CSF, GM-CSF, NGF-β and CCL2), or in a regular (“R”)microglial differentiation medium (comprising GM-CSF and IL-34, as usedin the Examples 1 and 2). Except where stated otherwise, the materialsand methods for this Example are the same as those described above forExamples 1 and 2.

In one exemplary protocol, the following steps are performed on theindicated days:

Day 0: Induce undifferentiated iPSC colonies grown onto Matrigel(roughly 1 mm in diameter) by switching to mTSeR Custom supplementedwith 80 ng/ml BMP4. Change media every day.

Day 4: Switch media to StemPro-34 with 2 mM Glutamax, 25 ng/ml bFGF, 100ng/ml SCF and 80 ng/ml VEGF.

Day 6: Switch media to StemPro-34 with 2 mM Glutamax, 50 ng/ml SCF, 50ng/ml IL-3, 5 ng/ml TPO, 50 ng/ml M-CSF, 50 ng/ml Flt-3.

Day 10: Collect the cells from the supernatant, spin-down and re-suspendin StemPro-34 with 2 mM Glutamax, 50 ng/ml SCF, 50 ng/ml IL-3, 5 ng/mlTPO, 50 ng/ml M-CSF, 50 ng/ml Flt-3. Put the cells back into their well.

Day 14: Collect the cells from the supernatant, spin-down and re-suspendin StemPro-34 with 2 mM Glutamax, 50 ng/ml M-CSF, 50 ng/ml Flt-3, 25ng/ml GM-CSF. Put the cells back into their well. Repeat every fourdays.

Day 24-52: FACS sort or use magnetic beads isolation for CD14+ cells.CD14+ cells are frozen in ProFreeze or 50% FBS/10% DMSO with 60%viability. The CD14+ progenitors are used in the followingdifferentiation step to produce microglial cells.

Following the above step, plate the CD14+ progenitor cells at a densityof 50K-100K cells/cm2 on tissue culture treated plastic. Feed the cellswith: either the alternative “A” media (i.e. RPMI-1640 supplemented with2 mM Glutamax, 10 ng/ml M-CSF, 10 ng/ml GM-CSF, 10 ng/ml NGF-β and 100ng/ml CCL-2), or the regular “R” media (i.e. RPMI-1640 supplemented with2 mM Glutamax, 10 ng/ml GM-CSF and 100 ng/ml IL-34). If media “A” isused, change medium every other day for 2 weeks. If media “R” is used,change the medium every four days for 2 weeks. This step will producemicroglial cells from the CD14+ progenitor cells.

It is not essential to isolate the CD14+ cells (using FACS or magneticbeads) prior to performing the microglial differentiation step. Forexample, in some embodiments, instead of isolating CD14+ cells, all ofthe cells in the supernatant are used for the subsequent microglialdifferentiation step. In addition, one of skill in the art willrecognize that certain modifications of these exemplary protocols (e.g.variations in the concentrations of agents and timing of media changes,etc.) can be used and are within the scope of the present invention.

FIG. 9 provides a schematic illustration of such an exemplary protocolin which the alternative “A” medium is used for the final microglialdifferentiation step. The cells produced using the alternative “A”medium for the final differentiation step express the microglial markersIBA1 and CD11c (as determined by immunofluorescence-staining; see FIG.10) as well as CD11b, CD11c, CX3CR1, P2RY12, CD45 (as determined byFACs).

Studies were performed to test the effects of using the “A” versus “R”microglial differentiation medium, and also to test the effects of usingvarious different substrates for plating of the microglial progenitors.Table 4 below shows the percentage (%) of total cells that expressed theindicated markers when the microglial progenitors were plated on theindicated substrates (i.e. plastic, laminin, fibrobectin or matrigel)and when cultured in either the “A” or “R” media. The microglialprogenitors can be plated on any suitable substrate, including any ofthe substrates shown and tested below. In some embodiments, including,but not limited to, those where adherent cells are desired (e.g. forperforming immunofluorescence studies) plastic is used. In someembodiments, including, but not limited to, those where cell adhesion isnot required, Matrigel is used.

TABLE 4 Effects of Differentiation Media and Substrate on MicroglialMarker Profiles CD11b CD11c CX3CR1 P2RY12 CD14 CD45 Plastic-A 85 89.915.2 14.9 51 99.9 Plastic-R 89.6 86.9 23.1 12.4 54.9 99.7 Laminin-A 72.686.5 30 27.9 67 99.8 Laminin-R 80 89 38.1 35 72.6 99.7 Fibronectin-A77.1 90 38.8 29.7 76.6 99.9 Fibronectin-R 86 80.8 20 15 66.2 99.8Matrigel-A 46.6 78.4 76.5 59.7 95.7 99.6 Matrigel-R 69.9 89.2 72.9 58.493.9 99.9

As shown in FIG. 11, the microglia cultured in the “A” medium (likethose cultured in the “R” medium) were able to phagocytose carboxylatedlatex beads. As shown in FIG. 12, the cytokines released by microgliacultured either in the “A” or the “R” medium were found to be verysimilar. It was also found that iPSC-derived microglia cultured onplastic or ultra-low attachment (ULA) plates demonstrated expressionprofiles that clustered together, and clustered together with humanprimary microglia, and are separate from peripheral blood-derivedmacrophages polarized as M(−), M(LPS,IFNγ), M(IL4,IL13) and M(IL10).

The foregoing description of the specific embodiments of the inventionwill fully reveal the general nature of the invention such that otherscan, without undue experimentation, apply knowledge that is within theordinary skill of those in the art to readily modify and/or adapt suchspecific embodiments for various applications without departing from thegeneral concept of the present invention. Therefore, such adaptationsand modifications are intended to be within the meaning and range ofequivalents of the disclosed embodiments, based on the teaching andguidance presented herein. It is to be understood that the phraseologyor terminology herein is for the purpose of description and not oflimitation, such that the terminology or phraseology of the presentspecification is to be interpreted by the skilled artisan in light ofthe teachings and guidance.

REFERENCE LIST

In addition to the documents cited in other sections of this disclosure,the following references may provide additional context. All of thereferences cited in this disclosure are hereby incorporated by referencein their entireties. In addition, any manufacturer's instructions orcatalogues for any products cited or mentioned herein are incorporatedby reference. Documents incorporated by reference into this text, or anyteachings therein, can be used in the practice of the present invention.Documents incorporated by reference into this text are not admitted tobe prior art.

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1. A method for generating microglial cells comprising: a) culturinghuman pluripotent stem cells under conditions that induce myeloiddifferentiation, leading to the generation of CD14+ and/or CX3CR1+microglial progenitor cells, and b) culturing the CD14+ and/or CX3CR1+microglial progenitor cells in either (i) a first microglialdifferentiation medium comprising IL-34, or (ii) a second microglialdifferentiation medium comprising M-CSF, thereby generating humanmicroglial cells. 2-20. (canceled)
 21. A method of treatment orprevention comprising administering microglial cells generated using themethod of claim 1 to a subject having, suspected of having, or at riskof developing a disease or disorder associated with a defect in ordeficiency of microglial cells or microglial progenitor cells.
 22. Themethod of claim 21, wherein the disease or disorder associated with adefect in or deficiency of microglial cells or microglial progenitorcells is selected from the group consisting of amyotrophic lateralsclerosis (ALS), Alzheimer's disease (AD), multiple sclerosis (MS),Parkinson's disease, Rett syndrome, diffuse leukoenchephalopathy withspheroids, hereditary diffuse leukoenchephalopathy with axonalspheroids, frontotemporal lobar degeneration (FTLD), familial FTLD,schizophrenia, and autism spectrum disorders.
 23. The method of claim27, wherein the microglial cells are generated from induced pluripotentstem (iPS) cells derived from somatic cells obtained from the subject.24. A method of identifying a compound useful in the treatment orprevention of a disease or disorder associated with a defect in ordeficiency of microglial cells, the method comprising: contacting amicroglial cell generated by the method of claim 1 with a candidatecompound, and determining whether the candidate compound improves thedefect in or deficiency of microglial cells.
 25. The method of claim 24,wherein the method is a high-throughput method.
 26. The method of claim21, wherein the subject is human.
 27. The method of claim 21, whereinthe microglial cells are autologous.
 28. The method of claim 21, whereinthe microglial cells are allogeneic.
 29. The method of claim 28, whereinthe microglial cells are derived from a donor individual having anMHC/HLA type that matches that of the subject.
 30. The method of claim22, wherein the disease or disorder associated with a defect in, ordeficiency of microglial cells or microglial progenitor cells isParkinson's disease.
 31. The method of claim 22, wherein the subject hasa GBA N370S mutation.